Neelakanta Ravindranath

Stem Cell Factor/c-kit Up-regulates Cyclin D3 and Promotes Cell Cycle Progression via the Phosphoinositide 3-Kinase/p70 S6 Kinase Pathway in Spermatogonia

Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-ζ, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation.

Telomere length in male germ cells is inversely correlated with telomerase activity.

Abstract Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.

Expression of Vascular Endothelial Growth Factor Receptors During Male Germ Cell Differentiation in the Mouse

Abstract Overexpression of vascular endothelial growth factor (VEGF) in the testis of transgenic mice induces infertility, suggesting a potential role for VEGF in the process of spermatogenesis. Spermatogenesis occurs within the confines of the seminiferous tubules, and the seminiferous epithelium lining these tubules consists of Sertoli cells and germ cells in various stages of maturation. We investigated the source of VEGF and VEGF-target cells within the seminiferous tubules of the normal mouse testis. Sections of testes fixed in Bouin solution and embedded in paraffin were subjected to immunofluorescent staining with specific antibodies against VEGF, and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Total RNA was extracted from isolated populations of Sertoli cells, type A spermatogonia, pachytene spermatocytes, and spermatids. Primer pairs specific for VEGF and its receptors were designed and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed. Immunofluorescent studies indicated that VEGF is strongly expressed in the cytoplasm of Sertoli cells. VEGFR-1 and VEGFR-2 were not expressed by the Sertoli cell. In contrast, a differential expression of VEGF receptors was observed in germ cells. Although VEGFR-2 was expressed in the cytoplasm of type A spermatogonia, VEGFR-1 was expressed in the acrosomal region of spermatids and spermatozoa. Pachytene spermatocytes did not exhibit any staining. Further, we examined the transcription of VEGF and its receptors by RT-PCR. VEGF was actively transcribed only in Sertoli cells. The transcription of VEGFR-2 was confined to type A spermatogonia. Interestingly, VEGFR-1 was transcribed both in pachytene spermatocytes and round spermatids. The mRNA expression of VEGFR-1 and VEGFR-2 in germ cells was inversely correlated during postnatal development of the mouse testis. Thus, VEGF may play a potential role in regulating the initial stages of the process of spermatogonial proliferation through VEGFR-2 and spermiogenesis through VEGFR-1.

Identification of a Murine Testis Complementary DNA Encoding a Homolog to Human A-Kinase Anchoring Protein-Associated Sperm Protein

Abstract Using differential display reverse transcriptase-polymerase chain reaction (RT-PCR) we have cloned a cDNA that encodes a putative peptide with homology to a recently reported A-kinase anchoring protein-associated protein (ASP) in human sperm. The mouse cDNA was 864 bases in length and encoded for a putative protein of 230 amino acids that had 90% amino acid similarity with the human ASP. The N terminal amino acid sequence had 65% similarity to the rat, mouse, and human protein kinase A regulatory type II sequences. Expression of the gene encoding this ASP was specific to testicular germ cells. Northern blot analysis of testis RNA from 5-, 15-, 25-, and 40-day-old mice showed expression of the ASP gene, but similar analyses of busulfan-treated germ cell-deficient mice failed to detect its expression. In addition, Northern blot analysis did not detect expression of the ASP mRNA in cultured Sertoli cells or cultured interstitial cells. Northern blot and RT-PCR analyses did not detect the ASP mRNA in mouse spleen, brain, liver, lung, heart, kidney, skeletal muscle, ovary, or Sertoli cells. In situ hybridization analysis localized the ASP mRNA to the germ cell compartment of the seminiferous tubules in the testis.

Regulation of c-fos mRNA expression in Sertoli cells by cyclic AMP, calcium, and protein kinase C mediated pathways

The role of second messenger pathways, cyclic AMP, calcium, and protein kinase C (PKC) in the transcriptional regulation of c-fos protooncogene expression in rat Sertoli cells was investigated. c-fos expression was monitored by Northern blot analysis. Although the action of FSH on Sertoli cells is considered to be mediated by CAMP, dibutyryl CAMP (dbcAMP), a potent membrane permeable analog of cAMP, induced much less c-fos mRNA expression than FSH (<50%) suggesting that additional cAMP-independent mechanisms may mediate the effect of FSH on c-fos. Specific intracellular inhibitors of PKC decreased c-fos induction in response to FSH by more than 50%. lonomycin, which increases intracellular free calcium concentration, induced c-fos expression significantly. These data demonstrate that Sertoli cell c-fos mRNA expression is under multifactorial regulation by CAMP, calcium, and PKC.

Isolation of rat ventral prostate basal and luminal epithelial cells by the STAPUT technique

The prostatic epithelium consists principally of basal epithelial cells, luminal epithelial cells, and neuroendocrine cells. Several studies support the concept that among basal cells, a subpopulation of stem cells resides which is capable of giving rise to other stem cells, basal epithelial cells, and also luminal epithelial cells and neuroendocrine cells. Other investigators suggest that luminal epithelial cells can also regenerate prostatic epithelium. Availability of pure populations of basal and luminal epithelial cells will aid in studies on defining the cellular pathways of differentiation during normal and pathological conditions. This study was designed to isolate and characterize pure populations of basal and luminal epithelial cells from adult rat ventral prostates.

Anatomical variant of the lateral pectoral nerve innervating the anterior portion of the deltoid muscle: A case report

The deltoid muscle is innervated by the axillary nerve. There is no collateral nerve supply described for this muscle. Palsy of the axillary nerve is common in shoulder trauma due to its close relationship to the surgical neck of humerus.

Telomerase Activity Is Lost During Male Germ Cell Differentiation

During embryonic development, male primordial germinal cells, which arise from the yolk sac, migrate to the bilateral gonadal ridges and proliferate (1). They interact with coelomic epithelial and mesenchymal cells to organize into testicular cords. The primordial germinal cells become gonocytes, the precursor cells of spermatogonia. The epithelial and mesenchymal cells derived from the mesonephros give rise to Sertoli cells, peritubular myoid cells, and interstitial cells of the testis (2). At birth, the testicular cords display gonocytes and undifferentiated Sertoli cells (3). Gonocytes proliferate and form type A spermatogonia during the early neonatal period (4). The unique process of morphological and functional differentiation of type A spermatogonia into spermatozoa is termed spermatogenesis. Unlike many other cells in the body that only undergo functional differentiation, type A spermatogonia also undergo morphological differentiation. The important features of type A spermatogonia are (i) they can proliferate; (ii) they can renew and self-maintain their numbers; (iii) they can produce a large number of functional progeny, i.e., the differentiated germ cells; and (iv) they can regenerate the advanced genu cell types after injury. These features are the hallmarks of “stem cells” (5). Thus, type A spermatogonia could be considered as the “stem cells of the germ cell lineage” (6).